SumlTlaryInterleukin (IL) 12 is a proinflammatory cytokine produced by phagocytic cells, B cells, and other antigen-presenting ceils that modulates adaptive immune responses by favoring the generation ofT helper type 1 cells. IL-12 mediates some of its physiological activities by acting as a potent inducer of interferon (IFN) y production by T and natural killer cells. IFN-y enhances the ability of the phagocytic cells to produce IL-12 and other proinflammatory cytokines. Thus, IL-12-induced IFN-y acts in a positive feedback loop that represents an important amplifying mechanism in the inflammatory response to infections. We show here that IFN-~/ enhances IL-12 production mostly by priming phagocytic cells for lipopolysaccharide (LPS)-induced transcription of the IL-12 p40 gene, which encodes the heavy chain of the IL-12 heterodimer; furthermore, IFN-y directly induces transcription of the IL-12 p35 gene, which encodes the light chain of IL-12, and has at least an additive effect with LPS stimulation in inducing its transcription. The priming effect of IFN-y on the LPS-induced p40 gene transcription requires preincubation of the cells with IFN-~/for at least 8 h to obtain a maximal effect. The priming effect of IFN-~/for IL-12 production is predominantly at the transcriptional level for both the p40 and the p35 gene, and no evidence for a major role of posttranscriptional or translational mechanisms was found. A 3.3-kb human IL-12 p40 promoter construct transfected into cell lines recapitulated the tissue specificity of the endogenous gene, being silent in two human T cell lines, constitutively active in two human Epstein-Barr virus-positive B lymphoblastoid cell lines, and LPS inducible in the human THP-1 and mouse RAW264.7 monocytic cell lines. Because the RAW264.7 cell line is easily transfectable and regulates the endogenous IL-12 p40 gene in response to IFN-y or LPS similarly to human monocytes, it was used for analysis of the regulation of the cloned human IL-12 p40 promoter. A requirement for the region between -222 and -204 in both LPS responsiveness and IFN-'y priming was established. This region contains an ets consensus sequence that was shown to mediate activation of the promoter by IFN-y and LPS, as well as by a cotransfected ets-2. The -222 construct was also regulated in a tissue-specific manner. Two other elements, IRF-1 located at -730 to -719, and NF-IL6 at -520 to -512, were also studied by deletion analysis, which did not result in decreased response to IFN-y and LPS stimulation.

The Xenopus early-thymectomy model system is used to investigate the extent to which the thymus controls T-cell development and to probe the evolution of natural killer (NK) cells. Loss of T-cell function following thymectomy, together with the paucity of cells expressing monoclonal antibody-defined T-cell surface markers, and greatly reduced expression of T-cell receptor beta transcripts in spleen, liver and intestine, indicate that T-cell development in minimal in the absence of the thymus. Our findings therefore mitigate against the idea that a substantial extrathymic pathway of T-cell development exists in early vertebrate evolution. Rather, they suggest that in this amphibian representative T cells are predominately thymus dependent. In vitro studies with control and thymectomized Xenopus splenocytes reveal that a non-T/non-B population and also two T-cell subsets all display natural cytotoxicity towards allogeneic thymus lymphoid tumour cells (which are deficient in MHC antigen expression). Since Xenopus thymectomized early in larval development are permanently deficient in T cells, they may provide a useful phylogenetic model for the study of NK cells.

T cell receptor (TCR) genes have been identified in representatives of both cartilaginous and bony vertebrates. The CD3 chains that serve as signal transducing elements of the TCR complex in mammals have been defined to a limited extent in birds. In these studies a CD3 homologue was identified in an amphibian representative, Xenopus laevis, using degenerate oligomer primers designed from conserved regions of avian and mammalian CD3 gamma/delta subunits. The reverse transcriptase polymerase chain reaction amplified product of Xenopus splenocyte RNA was then used to isolate full-length cDNA clones from a splenic library. When employed as probes, the cDNA clones hybridized with a 1-kb mRNA transcript in Xenopus T cells, but not in other cell types. Comparison of the deduced amino acid sequence indicated a similar degree of homology with mammalian and avian CD3 gamma and delta chains. Genomic analysis indicated that the Xenopus CD3 molecule is encoded by five exons, a structure resembling the mammalian CD3 delta gene rather than the seven exon CD3 gamma gene. Southern blot analysis and sequencing of the 5' flanking region failed to yield evidence of a related Xenopus gene. This amphibian CD3 gene thus appears to represent an ancestral form of the mammalian CD3 gamma and delta genes.